11beta-hydroxylation of pregnadienes by coniothyrium helleborine



United States Patent (7 11B-HYDROXYLATION OF PREGNADIENES BYCONIOTHYRIUM HELLEBORINE Richard W. Thoma, Somerville, and John R. Gerkeand Josef Fried, New Brunswick, N. J., assignors to Olin MathiesonChemical Corporation, New York, N. Y., a corporation of Virginia NoDrawing. Application July 18, 1955, Serial No. 522,850

4 Claims. (Cl. 195-51) This application is a continuation-in-part of ourparent application, Serial No. 371,728, filed July 31, 1953.

This invention relates to a biosynthetic method for the preparation ofllfi-hydroxy-steroids of the pregnane (including the pregnene andpregnadiene) series, especially for the preparation of hydrocortisoneand A -pregnadiene-l1,8,17a,2l-triol-3,20-dione and 21-esters thereof,involving microbiological oxidation of the correspondingll-desoxy-steroid, especially 1l-desoxy-l7a-hydroxy-corticosterone (alsoknown as a -pregnene-l7a,21-diol-3,20- dione and, as hereinafterreferred to for brevity, as Compound S), A-pregnadiene-17a,21-diol-3,20-dione and 21-esters thereof.

More particularly, the method of this invention involves subjecting anll-desoxy-steroid of the pregnane series, especially Compound S, A-pregnadiene-17a,21- diol-3,20 dione or a 21-ester thereof to the actionof an enzyme (or enzymes, or enzyme system) of a microorganism of thegenus Coniothyrium (preferably Coniothyrium helleborine) in an aqueousmedium inthe presence of oxygen, and recovering the llfl-hydroxy-steroid(e. g., hydrocortisone, A -pregnadiene-l1/9,17a,2l-triol- 3,20-dione ora 2l-ester thereof) formed. The action of the enzyme can be utilizedeither by including the steroid in an aerated culture of themicroorganism in or on a suitable nutrient medium, or by bringingtogether in an aqueous medium the steroid, oxygen, and the enzyme ofnon-proliferating cells of the microorganism, the first alternativebeing preferred.

The ll-desoxy-steroids utilizable in the method of this inventioninclude, inter alia, Compound S (yielding hy drocortisone), progesterone(yielding llfi-hydroxyprogesterone), desoxycorticosterone (yieldingcorticosterone), 17oe-hydroxyprogesterone (yielding115,17a-dihydroxyprogesterone), and a -pregnadiene-17a,2l-diol-3,20-dione and 2l-esters thereof. Among the suitable esterifying acids, if a21-ester is used, are the organic carboxylic acids, particularly thehydrocarbon carboxylic acids having less than ten carbon atoms asexemplified by the lower alkanoic acids (e. g. acetic, propionic,butyric and manthic acids), the lower alkanedioic acids (e. g. succinicacid), the hydrocarbon aromatic acids (e. g. benzoic acid), and thehydrocarbon aralkanoic acids (e. g. phenylacetic and B-phenylpropionicacids).

A suitable nutrient medium essentially comprises a source of nitrogenousfactors and an assimilable source of carbon and energy. The latter maybe a carbohydrate (such as sucrose, molasses, glucose, maltose, starch,or dextrin), a fatty acid, a fat and/or the steroid itself. Preferably,however, the medium includes an assimilable source of carbon and energyin addition to the steroid. Among the fats utilizable for the purpose ofthis invention are: lard oil, soybean oil, linseed oil, cottonseed oil,peanut oil, coconut oil, corn oil, castor oil, sesame oil, 'crude palmoil, fancy mutton tallow, sperm oil, olive oil, tristearin, tripalmitin,triolein, and trilaurin. Among the fatty acids utilizable for thepurpose of this invention 2,793,163 Patented May 21, 1957 are: stearicacid, palmitic acid, oleic acid, linoleic acid, and myristic acid.

The source of nitrogenous factors may be organic (e. g. soybean meal,corn steep liquor, meat extract and/ or distillers solubles) orsynthetic (i. e. composed of simple, synthesizable organic and inorganiccompounds such as ammonium salts, alkali nitrates, amino acids or urea).

An adequate sterile-air supply should be maintained ice duringfermentation, for example, by the conventional methods of exposing alarge surface of the medium to air or by utilizing submerged aeratedculture.

The steroid may be added to the culture during the incubation period, orincluded in the medium prior to sterilization or inoculation. Thepreferred (but not limiting) range of concentration of the steroid inthe culture is about 0.01 to 0.10%. The culture period (or rather thetime of subjecting the steroid to the action of the enzyme) may varyconsiderably, the range of about 6 to 96 hours being feasible, but notlimiting.

The llfi-hydroxy-steroid (e. g., hydrocortisone) formed may be detectedand quantitatively measured without isolation by paper chromatography ofa concentrated extract of the culture filtrate, and (in the cases ofhydrocortisone and corticosterone) confirmed by rat glycogen depositionassay. The paper chromatography method, based on that of Zaifaroni andBurton U. Biol. Chem., 193, 749-67 (1951)], involves carefulstandardization based on hydrocortisone (for example) moving atone-third the rate of Compound S in the benzene-water system, and on itsmoving at four times the rate of 11- epi-hydrocortisone in abenzene-ethanol-water system. Thus, 8.0 ml. of culture filtrate isequilibriated with 5.0 ml. of methylisobutyl ketone (MIBK), 4.0 ml. ofthe MIBK phase is separated, evaporated to dryness and redissolved in0.20 ml. of a 1:1 mixture of chloroform and absolute ethanol. An amountof this chloroform-ethanol solution containing 10-1007 hydrocortisone isapplied to the paper strip, and the developed chromatogram is chartedwith the aid of an ultraviolet scanning device [Haines and Drake, Fed.Proc., 9, 180 (1950)]. The hydrocortisone zone is then cut out, elutedwith 10 ml. ethanol, and the ultraviolet absorption at 240 me isdetermined. Quantitative estimates are then made by reference to astandard curve prepared by adding several levels of hydrocortisone to8.0 ml. aliquots of unfermented nutrient medium, and carrying out theextraction.

The llfi-hydroxy steroid formed, may be recovered from the culture inwhich it is formed by extraction with an organic solvent (e. g. methylisobutyl ketone, chloroform and methylene dichloride), followed byconcentration of the extract and crystallization from a suitable organicsolvent.

The following examples are illustrative of the invention: EXAMPLE 1Water to make one liter.

The pH of the medium is adjusted to 7.0i-0.1 with 2 N NaOH solution, and50 ml. portions of the medium are distributed in 250 ml. Erlenmeyerflasks, and the flasks are plugged with cotton and sterilized byautoclaving for 30 minutes at 120 C. When cool, each of the flasks isinoculated with 5% of a spore suspension of Coniothyrium helleborine(obtainable, inter alia, from the Department of Botany, Kansas StateCollege and the American Type Culture Collection, Washington, D. 0,wherein it has been assigned the catalogue number 12,522). [Thesporulated culture is obtained by growing the microorganism on crackedcorn for two to three weeks to allow maximum sporulation to occur. Thesuspension is made preferably in 0.01% aqueous Duponal solution. 2.5 ml.of suspension, containing about one sixtieth of the spores from g. ofcorn, is used to inoculate 50 ml. of the first shaker flask stage] Theflasks are then mechanically shaken for 66 hours on a 280 cycle perminute rotary shaker at 25 C. and 6% by volume transfer is then made to40 flasks (250 ml.) containing 50 ml. portions of the following medium:

Water to make one liter.

Incubation is then carried out for 48 hours at 25 C. by mechanicallyshaking on a 280 cycle per minute rotary shaker, after which 500 mg. ofA -pregnadiene-17m2ldiol-3,20-dione is added in 20 ml. of methanol.Incubation is continued for another 48 hours. The contents of the flasksare pooled, filtered on a Seitz pad, and washed through with about 10%water. The volume of filtrate and wash is 1875 ml.

([1) Isolation of the A -pregnadiene-H/3J7a,21-tri0l- 3,20-zlione.-Thethus-obtained culture filtrate is extracted with 3 one-liter portions ofchloroform and the resulting chloroform solution evaporated to drynessin vacuo. The residue (about 238 mg.) is triturated with chloroform andthe resulting crystalline precipitate (about 146 mg.), which consists ofa mixture of starting material and the desired hydroxylated product, isacetylated with 2 ml. of anhydrous pyridine and 2 ml. of aceticanhydride at room temperature for 18 hours. The mixture of acetatesobtained by evaporation of the reagents is crystallized four times from95% alcohol, yielding pure A pregnadiene-l1B,17a,2l-triol-3,20-dioneZI-acetate, M. P. about 237239 C. [a] +112 (dioxane);

3.07 1, 5.72 2, 581a, 616a, 624a, 6.30 2 [which infrared spectrum isidentical with that of an authentic sample].

Saponification of the above 21-acetate with potassium carbonate inmethanol furnishes authentic A-pregnadiene-l15,17a,21-t1i-ol-3,20-dione, M. P. about 239240 C.

max.

EXAMPLE 2 (a) Fermentation.-A fermentation medium (A) of the followingcomposition is prepared:

Distilled water to make one liter.

[The same medium may be employed for germination of the inoculatingmicroorganism] The pH of the medium is adjusted to 7.0-' -0.1 with 2normal NaOH solution; 0.5 g. of Compound S is added; 100 ml. portions ofthe medium are distributed in 500 ml. Erlenmeyer flasks, and the flasksare plugged with cotton and sterilized by autoclaving for 30 minutes at120 C.; and when cool each of the flasks is inoculated with 5l0% of avegetative inoculum of Coniothyrium helleborine. [A stock culture of themicroorganism is obtainable from the Department of Botany, Kansas StateCollege and the American Type Orlture Collection, Washington, D. C.under number 12,522; and the inoculum is obtained by growing themicroorganism in a medium of the same composition for 48 hours.] Theflasks are then mechanically shaken for 72 hours in a room maintained at25 0., following which the contents of the flasks are pooled, adjustedto pH 4.0:02 with 12 N sulfuric acid, and filtered by suction throughSeitz filter pads to remove the mycelium.

(b) Isolation of the hydrocortisone f0rmed.--A quantity of culturefiltrate obtained as described in a by fermentation of 2.0 g. ofCompound S is extracted with twice its volume of chloroform, and thechloroform extract is filtered and evaporated to dryness in vacuo. Theresidue (total steroid fraction, weighing about 1.166 g.), is trituratedwith chloroform, leaving behind a crystalline residue weighing about 256mg. and melting at about 197-200 C., 244 mg. of this crystalline residueis acetylated with 1 ml. of acetic anhydride in 2 ml. of pyridine for 16hours at room temperature, and the acetylating reagents are removed inhigh vacuum. The residue (weighing about 268 mg.) is dissolved in 1 ml.of chloroform and 9 ml. of benzene, and the solution is chromatographedon 5 g. of silica gel. Elution with chloroform in benzene (1:1) yieldsfirst a lower melting fraction (l-192 C.; about 28 mg. in 75 ml.),followed by essentially pure ll-epi-F diacetate (about 97 mg. in about500 ml.). After recrystallization from acetone-hexane, the latter meltsat 223-225 C., shows no depression of melting point when mixed with anauthentic sample of ll-epi-F diacetate, and has an infrared spectrumidentical with that of the sample. Further elution of the column withchloroform yields an additional amount of ll-epi-F diacetate (about 37mg. in about 100 ml.), followed by a mixed fraction in the next 200 ml.of chloroform. Final elution with chloroform (about 500 ml.) andevaporation of the chloroform yields about 17 mg. of crystallinematerial, differing from the ll-epi-F diacetate by its low solubility inchloroform. Recrystallization of this finally eluted material fromacetone yields pure hydrocortisone acetate, having the followingproperties: M. P. 2l6-2l9 C.; [a] +156 (c., 0.32 in chloroform);

A 3; 241 m (e=16,700)

infrared spectrum identical with that of an authentic sample ofhydrocortisone acetate.

Hydrocortisone may be produced from its acetate thus obtained byconventional means, e. g., hydrolysis with dilute sodium bicarbonatesolution; and, if desired, the hydrocortisone may be converted intoesters of other acids.

EXAMPLE 3 The same fermentation conditions as described in section a,Example 2, are employed except the inoculum is a 48-hour culture of themicroorganism in fermentation medium A, the fermentation medium contains300 mg. of Compound S per 600 ml., and the culture is harvested at 67hours.

EXAMPLE 4 The same fermentation conditions as described in section a,Example 2, are employed, except that the Compound S is added to the24-hour culture, 100 mg. of this compound in 3.0 ml. of methanol beingadded to each 100 ml. of culture. A 10% conversion of the compound tohydrocortisone is obtained after 24 hours incubation of the medium.

EXAMPLE 5 Coniothyrizun helleborine is grown in fermentation medium A(inoculation being with a 48-hour growth in the same medium, originatingwith spores) for 48 hours;

then desoxycorticosterone (in methanol) is added to give a 0.025%desoxycortioosterone concentration (and 1% methanol concentration) inthe medium; and after 24 hours incubation, the culture is harvested andthe culture filtrate treated (e. g., by chloroform extraction andchromatographic fractionation) to recover the corticosterone (andll-epi-corticosterone) formed.

The invention may be otherwise variously embodied within the scope ofthe appended claims.

We claim:

1. The method of producing a steroid selected from the group consistingof A -pregnadiene-llfi,l7u,2l-triol-3, 20-dione and 21-esters thereof,which comprises subjecting a steroid selected from the group consistingof A pregnadiene-17a,21-diol-3,20-dione and ZI-esters thereof to theaction of an enzyme of Coniothyrz'um helleborine in an aqueous medium inthe presence of oxygen, and recovering the steroid formed.

2. The method of claim 1 wherein the steroid is Apregnadiene-l7a,21-diol-3,20-dione.

References Cited in the file of this patent UNITED STATES PATENTS2,602,769 Murray July 8, 1952 2,649,400 Murray Aug. 18, 1953 2,649,401Haines Aug. 18, 1953 2,649,402 Murray Aug. 18, 1953 2,658,023 Shull Nov.3, 1953

1. THE METHOD OF PRODUCING A STEROID SELECTED FROM THE GROUP CONSISTINGOF $1,4-PREGNADIENE-11B,17A,21-TRIOL-3, 20-DIONE AND 21-ESTERS THEREOF,WHICH COMPRISES SUBJECTING A STEROID SELECTED FROM THE GROUP CONSISTINGOF $1,1PREGNADIENE-17A,21-DIOL-3,20-DIONE AND 21-ESTERS THEREOF TO THEACTION OF AN ENZYME OF CONIOTHYRIUM HELLEBROINE IN AN AQUEOUS MEDIUM INTHE PRESENCE OF OXYGEN, AND RECOVERING THE STERIOD FORMED.